The Botulinum toxins (Bttxs) are among the most potent toxins to animals, e.g. the LD50 in mice is about 1 ng/kg. Bttxs comprise a family of seven distinct serotypes (A–G). Bttxs are composed of two subunits comprising a 100 kdal nerve-cell targeting heavy chain and a 50 kdal endoproteolytically active light chain. These toxins are Zn-metalloproteases and contain a Zn-protein binding motif HEXXH.
However, Zn-metalloprotease inhibitors, such as angiotensin converting enzyme inhibitors, captopril and phosphoramidon, are not effective inhibitors of Bttxs. Although Zn-chelators inhibit Bttx protease activity in vitro, they merely delay the protease activity in vivo and in tissue preparations comprising intact nerve and muscles cells and/or tissues. Furthermore, some Zn-chelators are toxic at concentrations necessary to delay the Bttx protease activity. Although dithiocarbamates inhibit other Zn-containing proteins such as SOD, they are ineffective against the Bttx serotype B (BttxB). Clearly, inhibitors of the various Bttx serotypes, such as BttxB, are needed.
BttxB specifically cleaves synaptobrevin (VAMP2) between glutamine 76 and phenylalanine 77 (QF bond or cleavage site). There is an obligatory requirement for a relatively long substrate for the in vivo target VAMP2 as shown by efforts to produce a minimum length substrate. It has been shown that 30 amino acids of VAMP2 are required and 40 amino acids of VAMP2 are required for optimum cleavage. See Shone, C. C. et al. (1993) Eur. J. Biochem. 217:965–971. V2, a peptide derived from VAMP2, is a sequence of 10 amino acids located 4 residues upstream from the cleavage site, and was found to inhibit Bttx activity. See Pellizzari R. et al. (1996) J. Biol. Chem. 271:20353–20358. In VAMP2, a mutation of the C-terminal amino acids had little effect; whereas a helix disrupting substitution of Pro for Ala inhibited BttxB activity by 28%. Further, replacement of several negatively charged amino acids led to almost complete inactivity. See Whitcome, M, et al. (1996) FEBS Let. 386:133–136).
Computer-aided secondary structure analysis of VAMP2 predicted two stretches of α-helical structure flanking the cleavage site QF. See Witcome, M. R. et al. (1996) FEBS Let. 386: 133–136. Computer-aided tertiary structure analysis indicates that the two helices could self associate to form a supersecondary structure of a helix bundle with the helices separated by a reverse turn. See Lebeda F. J., et al. (1996) Med. Defense Biosci. Rev. 204.
The above results indicate that more than just the QF bond is required to be recognized by the toxin for substrate cleavage.
We have previously described a new class of compounds, Buforinins, that have a characteristic conformation, a QF bond, and inhibit BttxB protease activity.
Recently, however, we have elucidated core structures present in these Buforinins and core structures exemplified by Substance P which may serve as the foundation structure or molecular building block of compounds which inhibit the protease activity of BttxB and Tttx. These core sequences may be the elemental structure of compounds which inhibit the protease activity of BttxB and Tttx. These core sequences and their uses are disclosed herein below.